Comparison of Nonviral Transfection and Adeno-Associated Viral Transduction on Cardiomyocytes

Abstract

Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and beta-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ). The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism. On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% beta-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity. We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.

Isolation and Culture of Neonatal Mouse Cardiomyocytes

“…: adenovirus, adeno-associated virus, lentivirus). Electroporation of neonatal cardiomyocytes is in our hands unsuitable for the delivery of DNA/RNA, however several reports show success using electroporation of embryonic and neonatal cardiomyocytes 38,39 . In a recent article Freezing of isolated neonatal cardiomyocytes for prolonged storage is extremely difficult and not recommendable due to low efficiency and low recovery rates.…”

Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds

“…However, non-viral vectors have not achieved the same levels of transgene expression (in a process termed transfection) as their viral counterparts, making vector design crucially important [7][8][9][10].…”

Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes

“…This synthetic mRNA could bypass innate anti-viral responses induced in human cell dedifferentiation and had kinetics substantially superior to established viral protocols (11)(12)(13). Furthermore, this method was non-mutagenic (2).…”